The long term goals of this project are to understand how Ndr kinase pathways are regulated and carry out their important roles in cell cycle progression, cytoskeletal organization, tumor suppression, and stem cell differentiation. Major rearrangements of the cytoskeleton are required for the transition between interphase and mitosis as well as undifferentiated to differentiated states. Failure to properly regulate cytoskeletal rearrangements can have profound effects including genomic instability, stem cell differentiation defects, cancer, and metastasis. Eukaryotic cells possess two conserved Ndr kinase pathways, called the SIN and MOR in fission yeast (S. pombe) or the Hippo/Lats and Ndr1/2 pathways in mammals. The two pathways are functionally distinct: the Hippo/Lats and SIN pathways play roles in cytokinesis and mitotic exit whereas the Ndr1/2 and MOR pathways function in cell polarity. In metazoans they have acquired additional functions in contact inhibition of growth, tumor suppression, and stem cell differentiation (Hippo/Lats), or growth promotion, chromosome congression and centrosome duplication (Ndr1/2). Despite their crucial functions in cell division, cell polarity, and development, few targets of these pathways are known. In Specific Aim 1, we will determine how signaling is coordinated between the MOR, Cdk1, and the SIN and whether this regulation is conserved in human cells. We will determine in Specific Aim 2 how newly identified targets of the SIN are regulated in late mitosis to promote cytokinesis, microtubule organization, and spindle checkpoint inactivation. In Specific Aim 3, we will identify substrates of the Hippo/Lats pathway and test hypotheses for how the pathway is activated in response to increased cell density to stop cell cycle progression. Overall these studies will show how the SIN promotes late mitotic events through substrate phosphorylation and crosstalk with the MOR pathway. Our studies on Lats2 targets and activation will have important implications for the processes of tumor suppression and tissue regeneration. PUBLIC HEALTH RELEVANCE: The pathways studied in this proposal have important functions in cell proliferation, cancer, and stem cell differentiation. This work will provide important insight into how these pathways work and how their activities are be regulated, which should lead to therapeutic approaches for cancer and tissue engineering. )